Enterochelin complexes

ABSTRACT

Complexes of enterochelin, and certain analogues thereof, with one of the metals indium and scandium are useful for the treatment of bacterial infections.

This is a divisional of application Ser. No. 35,415, filed May 2, 1979,now U.S. Pat. No. 4,269,850.

This invention relates to antibacterial agents.

The compound enterochelin is known to be an essential growth factor inrelation to the virulence of a variety of bacteria, for exampleEscherichia coli, due, it is believed, to the ability of this substanceto provide iron for the bacteria through competition with transferrin.It has now been found, however, that novelcomplexes of enterochelin withcertain metals exert a quite considerable antibacterial action. Theeffect is very specific, being limited to a small group of metals only.

Accordingly the present invention comprises a complex of enterochelin,or an analogue thereof as defined below, with one of the metals indiumand scandium.

Enterochelin is the cyclic trimer of 2,3-dihydroxybenzoyl-L-serine andhas the structure ##STR1## The present invention extends not only tocomplexes of the cyclic trimer but also to analogues thereof in which(a) the aliphatic ring system has been opened at one or more of theester linkages to give a linear form of the trimer, or the correspondingdimer or monomer, in which one or both of the resulting free carboxy andhydroxy groups may optionally be in derivative form; and/or (b) theserine has instead the D-configuration.

Among the complexes of the linear compounds, it is generally the casethat complexes of the trimerN,N',N"-tris(2,3-dihydroxybenzoyl)-O-seryl-O-serylserine are of moreinterest than complexes of the dimerN,N'-bis(2,3-dihydroxybenzoyl)-O-serylserine, which are in turn of moreinterest than those of the monomer N-(2,3-dihydroxybenzoyl)-serine. Suchgeneralisations are also broadly applicable to complexes of the linearcompounds in derivative form and when the serine is in the D- as well aswhen it is in the L-configuration, although the change from L- to D-configuration, for example in N- (2,3-dihydroxybenzoyl)-serine, mayenhance the value of a compound. Derivatives of these linear compoundsforming complexes according to the present invention include variouscommon forms of derivative described in the art for carboxy and hydroxygroups, i.e. esters and salts such as those with the physiologicallyacceptable ions mentioned below, and also those derivatives in whichdehydration has occurred at the aliphatic hydroxyl group to give adouble bond, for example with the formation in the case of the lineartrimer of the compoundN,N',N"-tris(2,3-dihydroxybenzoyl)-O-(α-aminoacrylyl)-O-serylserine.

In the case of enterochelin itself, the metals in the form of theirtrivalent cation are combined with the six phenolic hydroxyl groups togive an octahedral complex. It is believed that this complex has anegative charge of three units due to the occurrence of the conversion60H→60⁻ during the formation of the complex. It is preferred not to usethe complex in its acidic form and conveniently, therefore, the netcharge of the active anion is neutralised through the addition of afurther cation or cations, for example three sodium ions or other ionssuited to the context of antibacterial use of the complex, includingparticularly such additional physiologically acceptable ions aspotassium, ammonium, etc. Such neutralisation may conveniently beeffected, for example, by the use of aqueous sodium bicarbonate informulating the complex. The complexes formed with analogues ofenterochelin, as defined above, may also include additional ions whichneutralise the net charge of the analogue-metal combination arising in asimilar manner as just described and also in some cases from thepresence of a carboxy group.

Enterochelin itself is most readily obtained by the microbiologicalprocess described in the art, whilst the linear analogues may also beobtained by such a process or a process involving the hydrolysis ofenterochelin as described hereinafter, followed where applicable byappropriate derivitivisation. Analogues containing D-serine are preparedsynthetically starting from 2,3-dihydroxybenzoyl-D-serine or aderivative thereof and, as appropriate, effecting extension to two orthree such units, optionally followed by cyclisation, these stepsinvolving manipulative procedures known in the art.

The complexes are conveniently prepared by the reaction in a suitablemutual solvent of enterochelin or its analogue and a salt of theappropriate metal which provides the metal ions. Salts of particularinterest are those having a significant level of solubility in organicsolvents in view of the relatively low solubility (ca 1 mM) ofenterochelin in water, and for this reason the chlorides are preferredamong halide salts. Suitable solvents for the reaction include mixturesof ketones and alcohols e.g. acetone/methanol and ketone alone. Aqueousalcohols may be used for the more water soluble linear analogues.

The reaction usually takes place quite rapidly at room temperature and,depending upon the particular reactants and the solvent used,precipitation may occur from the reaction solution or, alternatively,the solvent may be removed in vacuo to leave the complex. The complexmay in either case, where desired, be washed with a suitable solvent,for example n-butanone followed by an ether such as diethyl ether, oralternatively some system such as n-butanone followed byn-butanone/glycerol which generally gives a material which is morereadily dispersible in aqueous media.

Among the enterochelin complexes, scandium enterochelin is generally themost active but is also somewhat less tolerated than the only slightlyless active inidium enterochelin. In the case of the complexes withanalogues such as N,N'N"-tris(2,3-dihydroxybenzoyl)-O-(α-aminoacrylyl)-O-serylserine, however, theindium complex may show the higher activity. Where desired, mixtures oftwo or more complexes according to the invention may be employed, andother active substances may be incorporated into the composition, forexample bacteriostatic substances such as kanamycin sulphate.

As indicated previously, the complex is conveniently formulated in aneutral form lacking an overall charge. The formulation of the complexfor use as a pharmaceutical for both human and animal administration maybe effected by a variety of methods, but usually involves the use of aphysiologically acceptable diluent or carrier. The complex may, forinstance, be applied as an aqueous suspension or emulsion for parenteraladministration, the composition therefore preferably being sterile andpyrogen-free. The complex may also be compounded for oral administrationin the presence of conventional solid carrier materials such as starch,lactose, dextrin and magnesium stearate. Alternative formulations are asaerosols, suppositories, cachets, and, for localised treatment, assuitable creams or drops.

The compositions may conveniently be formulated in unit dosage form,i.e. in the form of discrete portions each containing a unit dose, or amultiple or sub-multiple of a unit dose. Dosage levels may, however,vary quite considerably according to the particular type of treatment.The complexes are well tolerated so that, for example, mice willtolerate two doses per day each dose corresponding to 5.7 mg/Kg ofscandium enterochelin, for a period of at least 5 days and often forabout 11 days. In general, larger amounts are better tolerated as aseries of spaced doses as compared with one single dose of the sameamount, although single doses of the indium complex equivalent to 60mg/Kg have been administered to mice without apparent adverse effect.

The compositions are of interest for the treatment of a range ofbacterial infections, particularly those produced by gram negativebacteria and especially aerobic rather than anaerobic bacteria. Althoughsuch activity is not without certain exceptions, no activity having beenobserved against Pasteurella for example, it is nevertheless widespread,particularly among the Enterobacteriaceae. Thus activity has beenobserved against Escherichia, Pseudomonas and Klebsiella, Aerobacter andSalmonella for enterochelin complexes but those of linear analogues maybe more restricted in activity. As well as showing activity againstsystemic infections enterochelin complexes do inhibit the growth ofenteropathogenic E. coli in a medium containing the contents of therabbit small intestine. An interesting feature of this activity is itsenhancement by the presence of an iron chelator. The reason for thisenhancement is believed to be that in contrast to the situation inserum, free iron is usually available in the gut, and removal of thisfree iron by an iron chelator enables the scandium or indium complex tofunction more efficiently in the treatment of infections in the gut bydepletion of the supply of iron enterochelin competing for uptake.Conveniently, therefore, a composition according to the presentinvention may comprise an iron chelating agent in addition to thecomplex. A variety of physiologically acceptable iron chelating agentsmay be used and these may be of natural occurrence, for examplerhodotorulic acid or particularly desferal, although synthetic agentsnot of natural occurrence are preferred, such as diethylenetriaminepentaacetic acid (DTPA) and particularly ethylene diamine tetraaceticacid (EDTA), and especially ethylenediamine di-(O-hydroxyphenyl) aceticacid (EDDA). Such compositions containing an iron chelating agent are ofinterest particularly in the treatment of gastroenteritis caused by E.coli, typhoid fever, cholera caused by Vibrio cholerae and dysenterycaused by Shigella.

The invention is illustrated by the following Examples.

EXAMPLES Example 1: Preparation of Scandium Enterochelin Complex

Equal volumes of 0.01 M enterochelin (prepared essentially according tothe procedure of Rogers et al, Biochimica et Biophysica Acta, 1977, 497,548) and 0.01 M scandium chloride (ScCl₃.6H₂ O), both in n-butanone, aremixed at room temperature to give a white precipitate which is collectedby centrifugation. The precipitate is washed .sup.(1) twice withn-butanone and then once with ether; removal of the residual ethergiving the scandium enterochelin complex as an off-white powder which isrelatively insoluble in both aqueous and organic solvents.

Example 2: Preparation of Indium Enterochelin Complex

Equal volumes of 0.01 M enterochelin and 0.01 M indium chloride(InCl₃.3H₂ O), both in n-butanone, are mixed together and the solventremoved in vacuo to give the indium enterochelin complex. This isdispersible in saline containing 1% w/v sodium bicarbonate by means ofsonication.

Example 3: Preparation of Complex of Indium with N,N',N"-Tris(2,3-Dihydroxybenzoyl)-O-(α-Aminoacrylyl)-O-Serylserine

(A) Enterochelin (100 mg) is dissolved in 5.0 ml of acetone and thesolution is added to 10 ml of water under nitrogen. Controlled alkalinehydrolysis is then carried out at pH 11.0 and 22° C. with the aid of apH stat, the reaction being stopped by the careful addition of 1 Naqueous hydrochloric acid after 1.3 ester bonds have been hydrolysed.The resultant solution is evaporated to dryness in vacuo and the residuedissolved in 5.0 ml of water. After the addition of 0.10 ml of glacialacetic acid, the hydrolysate is fractionated by chromatography on a 3cm×40 cm column of 625 Sephadex (superfine) using 6% v/v aqueous aceticacid to equilibrate and elute the column; the 2,3-dihydroxybenzoylserine-containing compounds being detected by means of their adsorptionat 300 nm. The slowest running component, which gives a single spot onpaper chromatography, is isolated by extraction with ethyl acetate,washing, drying and evaporation to give a residue of N, N',N"-tris(2,3-dihydroxybenzoyl)-O-(α-aminoacrylyl)-O-serylserine .sup.(1).

(B) A 0.2 mM solution of the indiumN,N',N"-tris(2,3-dihydroxybenzoyl)-O-(α-aminoacrylyl)-O-serylserinecomplex .sup.(1) is prepared by mixing 0.10 ml of a solution of 2.36 mMof N,N',N"-tris (2,3-dihydroxybenzoyl)-O-(α-aminoacrylyl)-O-serylserinein ethanol with 0.049 ml of a solution of 5.0 mM In Cl₃ ·3H₂ O in 0.1 Naqueous hydrochloric acid, and then adding 0.96 ml of water andsufficient solid sodium carbonate to raise the pH to 7.0.

Example 4: In Vivo Tests of Bacteriostatic Activity of ScandiumEnterochelin Complex

Female TO mice weighing 23 to 25 grams are used in tests forantibacterial activity against three different types of bacteria, allinjections of the scandium complex being given by the intraperitonealroute as a dispersion in sterile saline containing 1% w/v sodiumbicarbonate.

(1) Pseudomonas aeruginosa

Twenty mice were each infected with 8.6×10⁴ of a mouse-virulent strain(approximately 8000 LD₅₀). One half of the animals served as untreatedcontrols whilst the other half received 0.2 ml of a 1.0 mM suspension ofscandium enterochelin complex at intervals of 1,6,24,48,55,72 and 79hours post-infection. The untreated controls had all died by 48 hourswhilst the treated animals remained healthy and were finally killed 5weeks later.

In a similar type of experiment where the animals were treated atintervals of 6,24,31,48 and 72 hours post-infection the survival ratewas 30%.

(2) Escherichia coli 0141 K85

Forty mice received 9×10⁶ E. coli 0141 (approx. 1 LD₁₀₀). One half ofthe group were given 0.2 ml 1.0 mM of scandium enterochelin complex atintervals of 1 and 6 hours post-infection. Ninety percent of theremaining untreated animals which served as controls had died by 32hours. Eighty percent of the treated animals survived without illeffects and were killed one month later.

(3) Klebsiella pneumoniae

Twenty mice were infected with 8.4×10⁴ bacteria (approx. 20 LD₅₀). Tenanimals were treated with 0.2 ml 1.0 mM of scandium enterochelin complexat intervals of 6,24,31,48,55,72 and 79 hours post-infection andremained healthy during the course of the treatment. The ten untreatedcontrols had died by the 5th day. The first death in the treated groupdid not occur until 8 days after infection. The survival rate of 20% wassimilar to that (40%) obtained when using kanamycin sulphate in the sametreatment schedule.

Example 5: In Vitro Tests of Bacteriostatic Activity of Indium andScandium Enterochelin Complexes Against E. coli Serotypes Associatedwith Gastroenteritis

Two alternative forms of medium are used. The fist medium (RIF) consistsof chyme from the rabbit small intestine mixed with medium 199 and 6%w/v aqueous NaHCO₃, and then diluted with saline, the proportions beingsuch as to give a final medium containing 10% v/v chyme, 0.2% w/v NaHCO₃and of one quarter strength in medium 199, having a pH of 7.5. Thesecond medium (TSB) which acts as a control is trypticase soy brothcontaining 0.6% w/v NaHCO₃. Each medium is stirred at 37° C. under anatmosphere of 5% CO₂, 85% N₂ and 10% O₂ (by volume). The media areinoculated with E. coli of serotype 0111, 0141 or 0149 using in eachcase an inoculum of ca. 10³ bacteria per ml. In certain cases one of theiron chelators ethylenediamine di-(O-hydroxyphenyl) acetic acid (EDDA),diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetra aceticacid (EDTA) and desferal (DF) is added prior to inoculation. In certaincases 10μM of the In³⁺ or Sc³⁺ complex of enterochelin is added 2 hoursafter inoculation. The levels of E. coli population in the variousmedium samples are determined at intervals by viable counts of dilutionson plates. The values of log₁₀ of viable counts at 7 hours afterinoculation are shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        E. coli          Iron    Enterochelin                                                                           log 10 viable                               serotype                                                                              Medium   Chelator                                                                              complex  count at 7 hours                            ______________________________________                                        0111    RIF      --      Sc       7.2                                         0111    RIF      EDDA    Sc       4.7                                         0141    RIF      EDDA    --       8.2                                         0141    RIF      EDDA    Sc       5.0                                         0149    RIF      EDDA    --       8.6                                         0149    RIF      EDDA    Sc       5.3                                         0141    RIF      DTPA    --       8.7                                         0141    RIF      DTPA    In       7.0                                         0141    RIF      EDTA    --       8.7                                         0141    RIF      EDTA    In       6.6                                         0141    RIF      EDDA    --       8.5                                         0141    RIF      --      In       8.5                                         0141    RIF      EDDA    In       5.5                                         0141    TSB      DF      --       8.5                                         0141    TSB      --      In       7.9                                         0141    TSB      DF      In       5.8                                         0141    TSB      DF      Sc       5.4                                         ______________________________________                                    

Example 6: In Vitro Tests of Bacteriostatic Activity of Complex ofIndium withN,N',N"-Tris(2,3-Dihydroxybenzoyl)-O-(α-Aminoacrylyl)-O-Serylserine

Rabbit plasma is inactivated by heating at 56° C. for 30 minutes andthen inoculated with Escherichia coli 0141 at 37° C. under an atmosphereof 5% CO₂ ; 85% N₂ and 10% O₂ (by volume). After 2 hours the indiumN,N',N"-tris(2,3-dihydroxybenzoyl)-O-(α-aminoacrylyl)-O-serylserinecomplex is added to a final concentration of 2 μM to one set of plasmasamples whilst a second set remains untreated as a control. The level ofE. coli population in the plasma samples are determined at inoculationand at 2,4,6 and 7 hourly intervals thereafter for both sets by viablecounts of dilutions on plates. The values of log₁₀ of viable counts areshown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                    log 10 Viable Counts                                              Time (hours)  Control  2 M In.sup.3+  complex                                 ______________________________________                                        0             2.71     2.71                                                   2             3.91     3.90                                                   4             4.63     4.20                                                   6             5.74     4.52                                                   7             6.38     4.53                                                   ______________________________________                                    

A similar in vitro experiment has shown activity for the indium complexof N,N'-bis(2,3-dihydroxybenzoyl)-O-serylserine against Klebsiella.

I claim:
 1. An indium or scandium complex of a compound containing atleast one serine moiety in its D- or L- configuration and selected fromthe group consisting of N,N',N"-tris(2,3-dihydroxybenzoyl)-O-seryl-O-serylserine, N,N'-bis(2,3-dihydroxybenzoyl)-O-serylserine, and derivatives of said compoundinvolving the carboxy and hydroxyl groups of said compound, thederivatives of said carboxy group being pharmaceutically acceptableesters and salts thereof and the derivatives of said hydroxyl groupbeing the resultant double bond from the dehydration of said compoundinvolving said hydroxyl group.
 2. The complex of claim 1, wherein saidcomplex is the indium complex.
 3. An antibacterial pharmaceuticalcomposition, comprising:an antibacterially effective amount of a complexof a compound containing at least one serine moiety in its D- or L-configuration and selected from the group consisting of N,N',N"-tris(2,3-dihydroxybenzoyl)-O-seryl-O-serylserine,N,N'-bis(2,3-dihydroxybenzoyl)-O-serylserine and derivatives of saidcompound involving the carboxy and hydroxyl groups of said compound, thederivatives of said carboxy group being pharmaceutically acceptableesters and salts thereof and the derivative of said hydroxyl group beingthe resultant double bond from the dehydration of said compoundinvolving said hydroxyl group, with indium or scandium.
 4. Thecomposition of claim 3, wherein said complex is the indium complex.
 5. Amethod of treating bacterial infections in a human or animal subject,comprising:administering to said subject an antibacterially effectiveamount of a complex of a compound containing at least one serine moietyin its D- or L- configuration and selected from the group consisting ofN,N',N"-tris(2,3-dihydroxybenzoyl)-O-seryl-O-serylserine, N,N'-bis(2,3-dihydroxybenzoyl)-O-serylserine and derivatives of said compoundinvolving the carboxy and hydroxyl groups of said compound, thederivatives of said carboxy group being pharmaceutically acceptableesters and salts thereof and the derivative of said hydroxyl group beingthe resultant double bond from the dehydration of said compoundinvolving said hydroxyl group, with indium or scandium.
 6. The method ofclaim 5, wherein said complex is the indium complex.
 7. The compositionof claim 3, which additionally comprises a physiologically acceptableiron chelating agent.
 8. The composition of claim 7, wherein said agentis not one of natural occurrence.
 9. The composition of claim 8, whereinsaid agent is diethylene triamine pentaacetic acid, ethylene diaminetetraacetic acid or ethylene diamine di-(O-hydroxyphenyl) acetic acid.10. The composition of claim 9, wherein the agent is ethylene diaminedi-(O-hydroxyphenyl) acetic acid.